The present invention relates to methods for the treatment of inflammation, inhibition of cyclooxygenase-2 gene transcription, and in vivo activities of caffeic acid derivatives.
Unlike cyclooxygenase-1 (COX-1), an enzyme that has constitutive expression in many tissues, cyclooxygenase-2 (COX-2) is induced by inflammatory stimuli like cytokines [1], lipopolysaccharide [2, 3], and mitogens [4, 5]. Both enzymes convert arachidonic acid to prostaglandin E2 (PGE2) and show similar kinetics for converting arachidonic acid to prostaglandin G2 and prostaglandin H2[6], but each appears to have different selectivity for non-steroidal antiinflammatory agents. With the recent development of specific COX-2 inhibitors, investigators have been able to more precisely define the roles of COX-1 and COX-2 enzymes in biological systems.
Cytokine-mediated COX-2 gene activation in pancreatic xcex2-cells inhibits glucose stimulated insulin secretion through the generation of PGE2. The role of COX-2 gene activation and PGE2 production in the context of cellular stress may be viewed from two perspectives. First, PGE2 production is an attempt by the pancreatic xcex2-cells to preserve function during an immune inflammatory attack. By generating PGE2, the xcex2-cell sets off a regulatory mechanism to limit glucose stimulated insulin secretion that in turn allows the xcex2-cell to conserve ATP and utilize its energy stores to transcribe genes that confer protection against oxidative stress. In addition, PGE2 release from xcex2-cells may bias the immune towards a Th2 profile. PGE2 stimulates Th2 cytokine production in human lymphocytes [15], inhibits LPS-induced IL-1xcex2 production in microglial cells [3], limits Th1 cytokine responses [16], and prevents generation of interferon-xcex3 by inhibiting human IL-12 production [17]. Alternatively, PGE2 acts as a proinflammatory agent by inducing leukocyte migration [18], endothelial adhesion [19], painful response [20], and antigen stimulated interferon-xcex3 production in Th1 lymphocytes [21]. These apparent conflicting actions of PGE2 may ultimately be related to the ambient concentration of PGE2 in the affected tissue. Low concentrations of PGE2 as seen during basal states may promote cell survival, while high concentrations of PGE2 may promote cell demise. In this context, it is interesting to note that both COX-1 and COX-2 have endogenous peroxidase activity that may also contribute to either a pro- or anti-inflammatory state [6].
Pancreatic islets, like other tissues, express low basal levels of COX-2 and its metabolic end product, PGE2. Cellular stress can increase COX-2 mRNA levels from 3 to 5-fold and PGE2 production by greater than 100-fold. Early studies demonstrated that PGE2 production could be stimulated with alpha-adrenergic agonists and that prostaglandin synthase inhibitors could reverse the alpha-adrenergic-mediated inhibition of glucose-stimulated insulin secretion in human subjects [22]. Further work revealed that PGE2 had no effect as an insulin secretagogue, but did inhibit glucose-stimulated insulin secretion in pancreatic xcex2-cells [8, 9]. PGE2 mediates its inhibitory effect on glucose-stimulated insulin secretion through stimulation of a pertussis-toxin sensitive GTPase protein that has yet to be cloned, but is likely to reside in the insulin secretory granule [23].
These findings led to the hypothesis that PGE2 may mediate the inhibitory effect of interleukin-1xcex2 (IL-1xcex2) on glucose-stimulated insulin secretion, since IL-1xcex2, itself, increases PGE2 production. However, McDaniel and colleagues demonstrated that nitric oxide (NO), not PGE2, mediates the inhibitory effects of IL-L1xcex2 on glucose-stimulated insulin secretion in rat pancreatic islets [24]. In addition, Turk and colleagues showed that L-NMMA, an inhibitor of iNOS, abrogated the inhibitory effect of IL-1xcex2 on glucose stimulated insulin secretion and partially inhibited PGE2 production through a mechanism that is likely to be post-translational (i.e., NO increases arachidonic acid substrate availability by inhibiting the reacylation of arachidonic acid into membrane phospholipid [25]). Thus, NO led to higher intracellular levels of arachidonic acid that could be converted to 12-HETE by 12-LO, and theoretically, PGE2 by COX-2, since arachidonic acid is a substrate for both 12-LO and COX-2. These results support prior studies in human islets showing that indomethacin, a non-selective cyclooxygenase inhibitor, enhanced glucose stimulated insulin secretion [26]. By increasing ambient arachidonic acid levels in the xcex2-cell, indomethacin enhanced glucose stimulated insulin secretion since arachidonic acid, itself, is a potent insulin secretagogue [27]. In addition, by blocking PGE2 production through the inactivation of COX-1 and COX-2, indomethacin prevented PGE2 mediated inhibition of glucose stimulated insulin secretion.
More recently, Robertson demonstrated that the selective COX-2 inhibitor NS-398 partially restored glucose-stimulated insulin secretion in HIT cells treated with IL-1xcex2 for 24 hours (Diabetes 1999; 48:Supp. 1 :A1017). The implication of this study is that PGE2 may participate in cytokine-mediated pancreatic xcex2-cell dysfunction, although this hypothesis has yet to be formally proven.
The selective COX-2 inhibitors discovered to date work at the post-transcriptional level. Callejas, et al. (1999) [30], for example, reported that indomethicin and the COX-2 specific inhibitor NS398, while both suppressing the activity of COX-2, lead to an accumulation of the protein in primary cultures of fetal hepatocytes and in cultured peritoneal macrophages. The authors found that the increased COX-2 levels were not the result of increased mRNA production, postulating that the accumulation was due to post-translation effects, such as increased stabilization of the enzyme, or decrease in the synthesis of prostaglandins that favor COX-2 degradation, or both. Callejas, et al. at 1240. Chan, et al. (1999) [31] obtained similar results with the selective COX-2 inhibitor rofecoxib in human osteosarcoma cells and Chinese hamster ovary cells. Chan, et al. found their results to be consistent with a two-step time-dependant model of reversible enzyme inhibition involving the formation of a tightly bound 1:1 enzyme-inhibitor complex. Chan, et al. at 555, 557. Gierse, et al. (1999) [32] conducted an in vitro comparison of the selective COX-2 inhibitor celecoxib and several non-steroidal anti-inflammatory drugs (NSAIDs), and proposed at least four distinct mechanisms of COX-2 inhibition, all of them at the post-translational level: (i) competitive; (ii) tight binding, time-dependent; (iii) weak binding, mixed; and (iv) covalent binding. Gierse, et al. at 615. In a recent minireview of research on the mechanisms of COX-1 and COX-2 catalysis and inhibition, Marnett, et al. (1999) [33] acknowledged that the regulatory aspects of cyclooxygenase function where poorly-understood (for example, the reason for the existence of two distinct cyclooxygenase genesxe2x80x94COX-1 and COX-2xe2x80x94sometimes expressed in the same cell type, is not known). Marnett, et al. at 22906. The authors described the topic as xe2x80x9can extremely important and exciting area of investigation.xe2x80x9d Id. Furthermore, Michaluart et al. [35] investigated the effect of caffeic acid phenethyl ester (CAPE) on COX-2 activity and expression. Using both in vitro and in vivo models they demonstrated that this caffeic acid derivative inhibited the enzyme activity of both COX-1 and COX-2 at low concentration (14-28 xcexcM), while higher concentrations (35-70 xcexcM) inhibited COX-2 gene transcription. In addition, they utilized the rat carrageenan air pouch model (a standard model for evaluating anti-inflammatory properties of various drugs) to demonstrate that CAPE inhibited prostaglandin synthesis in a dose-dependent fashion (10-100 mg/kg).
Kirchner, et al. [36] utilized a dual COX-2/5-lipoxygenase inhibitor (RWJ63556) to inhibit a localized inflammatory response in a canine model. Oral administration of RWJ63556 at a dose of 3 mg/kg lead to significant sustained inhibition of leukocyte influx and prostaglandin synthesis over a 24 hour period, and compared to other COX-1/COX-2 inhibitors like dexamethasone and indomethacin.
Pancreatic xcex2-cells express low levels of COX-1 mRNA and somewhat higher levels of COX-2 mRNA when cytokines are not present [7]. Upon stimulation with cytokines like IL-1xcex2, COX-2 mRNA increases several fold, while COX-1 mRNA expression remains unchanged. Prior studies demonstrated that PGE2, the metabolic end product of cyclooxygenase activity, inhibits glucose-stimulated insulin secretion in rat islets [8, 9] and these observations led to the hypothesis that cytokine-induced pancreatic xcex2-cell cytotoxicity may be, in part, due to excessive PGE2 production.
It has been demonstrated recently that 12-lipoxygenase (12-LO) knockout mice are resistant to streptozotocin-induced diabetes [10] and leading to the hypothesis that part of this cytoprotective effect resided in the pancreatic xcex2-cell, since 12-LO is preferentially expressed in xcex2-cells compared to xcex1- and xcex2-cells [11]. Moreover, 12-LO knockout mice demonstrated no cytokine-inducible conversion of arachidonic acid to 12-hydroxyeicosatetraenoic acid (12-HETE) as expected, implying that 12-HETE generation may be cytotoxic to pancreatic xcex2-cells. Prior studies have demonstrated that arachidonic acid microparticles could induce COX-2 gene expression [12], raising the possibility that the 12-LO pathway product 12-HETE might also affect COX-2.
Certain caffeic acid derivatives have been found to be potent inhibitors of 12-LO. Cho, et al. (1991) [34]. This lipoxygenase is know to be implicated in inflammation and atherosclerosis through its production of the arachidonic acid metabolite 12-HETE. However, in vitro studies specifically showed that these derivatives did not inhibit cyclooxygenase activity. Cho, et al. at 1505.
It has now been found that lipoxygenase inhibitors do, in fact inhibit COX-2 activity, but by acting at the transcriptional level and suppressing expression of the COX-2 gene.
The present invention relates to methods of treating inflammation comprising administering a COX-2 transcription-inhibiting amount of a caffeic acid derivative, preferably a cyanocinnamate. In a preferred embodiment, cinnamamyl-3,4-dihydroxy-xcex1-cyanocinnamate is used. It has been found that this class of compounds, known in the art as 12-lipoxygenase inhibitors, have potent in vivo COX-2 inhibiting effect, unlike other classes of lipoxygenase inhibitors. The inhibitory effect of the caffeic acid derivatives on COX-2 is believed to occur at the transcriptional level, which may be one explanation for why the effect was not observed in in vitro enzyme assays such as those reported by Cho, et al. [34]
The COX-2 gene and 12-lipoxygenase (12-LO) gene are preferentially expressed over other isoforms of cyclooxygenase and lipoxygenase in pancreatic xcex2-cells. Inhibition of either COX-2 or 12-LO can prevent cytokine-induced pancreatic xcex2-cell dysfunction as determined by glucose-stimulated insulin secretion. It has been found that 12-HETE significantly increases COX-2 gene expression and prostaglandin E2 production, while a closely related lipid 15-hydroxyeicosatetraenoic acid (15-HETE), does not. However, interleukin-1xcex2 stimulated prostaglandin E2 production is completely inhibited by the preferential lipoxygenase inhibitor cinnamamyl-3,4-dihydroxy-xcex1-cyanocinnamate (CDC), a caffeic acid derivative. Finally, IL-1xcex2 has been found to fail to stimulate prostaglandin E2 production in 12-LO knockout islets, while C57BL/6 islets showed an 8-fold increase.
These data demonstrate not only that 12-HETE mediates cytokine-induced COX-2 gene transcription and prostaglandin E2 production in pancreatic xcex2-cells, but that caffeic acid derivatives, and in particular cynanocinnamates, can inhibit COX-2 activity by suppressing COX-2 gene expression.
FIG. 1: Western immunoblot of COX-2 protein expression in RIN m5F cells. 12-HETE (1-10 nM) shows equipotent COX-2 protein stimulation compared to IL-1xcex2 (0.3 ng/ml) at 24 hours. Lanes 1 and 2 demonstrate control COX-2 levels. Lanes 3 and 4 show IL-1 xcex2 stimulation. Lanes 5 and 6 show 12-HETE 1 nM stimulation and lanes 7 and 8 show 12-HETE 10 nM stimulation. Shown is one representative blot that was repeated twice.
FIG. 2: Western immunoblot of cultured intact porcine pancreatic islets. Islets were exposed to the indicated agents for 24 hours prior to protein extraction. IL-1 xcex2 (0.3 ng/ml) stimulated a 3-fold increase in COX-2 protein that was completely inhibited by CDC (11, M). CDC alone did not appreciably affect basal COX-2 protein levels.
FIG. 3: Semi-quantitative RT-PCR analysis of serum, IL-1 xcex2, and 12-HETE induced COX-2 gene expression.
Serum and 12-HETE induced a 3-fold increase in COX-2 mRNA expression, while IL-1 xcex2 induced a 2-fold increase. Shown is one representative autoradiograph out of two replicate experiments
FIG. 4: Semi-quantitative RT-PCR analysis of serum, IL-1 xcex2, and 15-HETE induced COX-2 gene expression.
Serum induced a 2-fold increase in COX-2 mRNA expression, while IL-1 xcex2 and 15-HETE induced a 1.5-fold increase. Shown is one representative autoradiograph out of two replicate experiments.
FIG. 5: Semi-quantitative RT-PCR analysis of serum, IL-1 xcex2, and 12-HETE induced COX-1 gene expression. No appreciable difference in COX-1 mRNA expression is noted.
FIG. 6: PGE2 production in RIN m5F cells. PGE2 was measured from conditioned culture medium 24 hours after the addition of indicated agents. 12-HETE induced a 10-fold greater increase in PGE2 than 15-HETE. Shown are 3-4 individual experiments per condition.
FIG. 7: CDC inhibits IL-1 xcex2 stimulated PGE2 production in RIN m5F cells. Preferential lipoxygenase pathway inhibitor CDC (luM) induced a statistically significant decrease in IL-1xcex2 induced PGE2 production as shown (*p less than .01). Shown are four separate experiments for each condition.
FIG. 8: PGE2 production in islets from 12-LO knockout mice and C57BL/6 mice. 12-LO knockout mice show no appreciable PGE2 production upon stimulation with IL-1 xcex2. In marked contrast, C57BL/6 mice show 7-fold increase in PGE2 production with IL-1xcex2 stimulation (*p less than .01). Shown are 3-4 separate experiments for each condition.
FIG. 9: Schematic diagram depicting signaling cascade leading from IL-1 xcex2 induced 12-LO activation to COX-2 gene transcription.